Use of urea-adjuvated polypeptides for diagnosis, prophylaxis and treatment

ABSTRACT

The invention relates to a method for infiltration of polypeptides in cells. The invention further relates to the use of the cells and urea-adjuvated polypeptides for the diagnosis, treatment or prevention of diseases. The invention further relates to the detection of polypeptide-specific immune cells.

The invention relates to a method for infiltration of polypeptides incells. The invention further relates to the use of the cells andurea-adjuvated polypeptides for the diagnosis, treatment or preventionof diseases. The invention further relates to the detection ofpolypeptide-specific immune cells.

The acquired branch of the immune system consists of a humoral (immuneglobulins) and a cellular immune defence.

Cellular and pathogen-specific polypeptides are processed fromantigen-presenting cells (APC) by specific cleavage and fragments(epitopes) thereof are presented together with MHC molecules of classesI and/or II on the cell surface. By means of their T-cell receptor, Tcells specifically recognise epitopes presented in the complex with theendogenous MHC proteins and start an immune reaction.

T cells can be subdivided into different effector populations usingspecific surface proteins. CD4⁺CD8⁻ T-helper cells are of centralimportance in controlling the immune defence. According to a specificrecognition of epitopes which are presented to them on the surface ofAPC together with MHC proteins, by secreting different messengersubstances (for example, cytokines) they regulate the production ofantibodies by B cells (humoral branch of the immune response) and theactivation of CD4⁻CD8⁺ cytotoxic T cells (CTL) (cellular branch of theimmune response).

The importance of CD4⁻CD8⁺ CTL lies in the recognition and destructionof cells and tissue which have degenerated and are affected bymicro-organisms or parasites. T cells are thus an important protectionmechanism of the acquired immune system for the prevention and controlof microbial, especially virus-induced diseases, and for the recognitionand destruction of degenerated endogenous cells. In addition to these Tcell populations, another population of circulating T cells has beendescribed by various working groups, which has a double positiveCD4⁺CD8^(dim) phenotype.

CD4⁺CD8^(dim) T cells express CD8αα homodimers and can be detected witha lower frequency (less than 2% of the total population of CD3⁺ T cells)in the blood. A transient or persistent expansion of CD4⁺CD8^(dim) Tcells was observed both in healthy persons and also in probands withvarious diseases, including infections with different viruses, forexample, the human immune deficiency virus type 1 (HIV-1) and the humancytomegalovirus (HCMV) as well as patients with various autoimmunediseases.

Furthermore, other populations of antigen-specific T cells, theso-called CD56⁺CD8⁺ and CD56⁻CD57⁺CD8⁺ NKT cells have been described.These cell populations express both a T cell receptor and also classicalNK cell markers and can also be detected with lower frequency (2 to 5%or 5 to 10%) in peripheral blood mononuclear cells (PBMC). So far, onlyvery little is known about the importance of these cell populations inthe control of microbial infections and tumours.

Professional APC such as dendritic cells, monocytes, macrophages butalso non-professional APC such as B cells play a central role both inthe triggering of a T cell response to exogenous immunogens and in theinduction of a T cell tolerance to endogenous tissue. The activation andproliferation of T cells takes place by the simultaneous triggering oftwo signals. The first signal is guided into the T cell by the T cellreceptor which recognises the epitope in association with MHC on thesurface of the APC.

The co-stimulatory signal is mediated by the specific interaction of theco-stimulatory molecules B7.1 (CD80) or B7.2 (CD86) on the APC with therelevant receptor (CD28) on the surface of the T cell. In the absence ofthe co-stimulatory signal, the epitope-specific T cell becomes anergic.Anergy describes a state in which the T cells cannot multiply and cannotrespond to an antigen.

The condition of a polypeptide decisively determines the efficiency androute of the epitope processing and presentation by an APC. In addition,the degree of activation of an APC and thus the profile of the inducedimmune response is adversely influenced by the form of administration ofa polypeptide. Thus, the concentration and biochemical properties of apolypeptide as well as the presence or absence of immune-modulatorysubstances (especially bacterial components such as lipopolysaccharides(LPS), nucleic acids (CpG-containing DNA) and polypeptides (e.g.flagellin)) are determining factors as to whether the cellular (Thelper-1 (Th-1)-type mediated immunity) or humoral branch (T helper-2(Th-2)-type mediated immune response) of the immune system is activatedor whether the immune response proceeds tolerogenically.

Hitherto, only a very few methods for the incorporation of polypeptidesinto mammalian cells had been described.

Hitherto, for example, mechanical methods of microinjection andelectroporation had been used with varying success for transferringprotein into cells (Mi et al. (2000); Mol. Ther. 2:339; Schwarze et al.(2000); Trends Cell Biol. 10:290). Other methods for polypeptidetransfer into cells are based on using protein transduction domains(PTD).

These amino acid sequences comprising 10 to 35 amino acids originate forexample from the HIV Tat protein, the Herpes Simplex Virus (HSV) VP22protein or Antennapedia. In addition, synthetic PTD sequences weredetermined by means of phage libraries. The membrane prevalence ofpolypeptides can be increased considerably by their coupling of thesewith PTD. Other methods described for protein transfer into cells arebased on using various cationic lipid formulations or the incorporationof polypeptides in ISCOM® particles (CSL Limited, Victoria, Australia).All these methods are too work- or cost-intensive for routine use. Inaddition, many of the particular transfer systems possess cytotoxic (forexample, liposomes) or modulatory properties (ISCOM® particles) whichcan subsequently adversely influence the natural properties of thetreated cells.

Bearing in mind the importance of the cellular immune response forcontrolling microbial infections and tumours, many new strategies forthe in vivo induction of epitope presentation on MHC class I and IIproteins in immune cells are currently being tested. These include theuse of (lipo-) peptides, (lipo-) proteins, particular immunogens, livingattenuated bacteria and viruses, recombinant living vaccines (based onvarious recombinant bacteria and viruses) and DNA vaccines. Furthermore,ex vivo treated autologous APC which present specific peptides in thecontext with MHC proteins of classes I and II are a suitable reagent forthe induction of polypeptide-specific immune responses, especially intherapeutic treatments. In earlier studies, APC pulsed with tumourextracts or cell lysates have proved suitable for simultaneouslyinducing CD4⁺ and CD8⁺ T cell responses (Herr et al. (2000), Blood,96:1857).

At the present time, various methods are available for stimulatingvarious populations of immune cells which are suitable to differentextents for detecting specific populations of antigen-specific immunecells.

Direct loading of membrane-bound MHC proteins with peptides of definedlength (optimally 8-11 amino acids for loading MHC class I proteins andoptimally 10 to 20 amino acids for loading MHC class II proteins) is amethod frequently used for stimulating defined populations of immunecells, especially CD8⁺ cytotoxic cells (CTL) and CD4⁺ T helper cells.However, important restrictions on the use of this stimulation methodfor the simultaneous measurement of different populations of immunecells lie in the fact that peptides of different size are specificallypresented on MHC proteins of classes I or II whereby, when using definedpeptides, it is not possible to simultaneously determine CD4⁺ T helpercells and CD8⁺ cytotoxic T cells. In addition, the specific recognitionof T cell epitopes is subjected to an MHC restriction; that is, personswho express different MHC proteins recognise different epitopes within apolypeptide which makes the analysis of polypeptide-specific T cells inprobands with variable MHC patterns considerably more difficult. Thus,only T cells which are directed against known epitopes in the contextwith defined MHC proteins can be specifically registered using thismethod.

In contrast, polypeptides produced recombinantly using various bacteriaas well as insect, yeast or mammalian cells are suitable for detectingpolypeptide-specific CD4⁺ T helper cells regardless of the MHCrestriction of the donor and the detailed knowledge of the T cellepitope localised in a polypeptide. However, recombinant polypeptidesare almost exclusively taken up and recovered via the MHC class IIprocessing and presentation route in APC so that this method isexclusively suitable for detecting CD4⁺ T helper cells.

Furthermore, various methods for denaturing polypeptides have also beendescribed which make it possible to supply these polypeptides to the MHCclass I and MHC class II processing and presentation route. Thesemethods include, for example, treatment of polypeptides with heat orsodium dodecyl sulphate (SDS). These methods proved to be suitable forachieving an epitope presentation on MHC class I and II molecules inmurine APC (Schirmbeck et al. (1994), Eur. J. Immunol., 24:2068);(Schirmbeck et al. (1995), Vaccine, 13:857). In these studies it wasshown that proteins denatured in various ways are taken up into the APCby means of various mechanisms and differ in terms of their efficiencyfor inducing a polypeptide loading of MHC class I polypeptides. Thus,compared with SDS-treated proteins, polypeptides treated using the heatinactivation method (1 hour at 60° C. or 15 min at 100° C.) only induceda slight stimulation of epitope presentation on MHC class I proteins intreated murine APCs. On the other hand, the SDS denaturing method provedto be little suited for use in human cell cultures because of the hightoxicity.

Another method for stimulating the MHC class I and II presentation ofepitopes on APC is based on the incorporation of polypeptides inparticular structures, for example, liposomes, particular carriersubstances, virus-like particles or lipoprotein particles. The firststudies confirmed the suitability of HIV-1 Pr55^(gag) virus-likeparticles for the diagnosis of CD4⁺ T helper cells and CTL (Sester etal. (2000), AIDS, 14:2653-60). However, the production of particle-boundpolypeptides is expensive and costly. Furthermore, these antigens arenot suitable for the diagnosis of other immune cell populations, forexample CD4⁺CD8^(dim) cytotoxic T cells, CD56⁺CD8⁺ and CD56⁻CD57⁺CD8⁺NKT cells.

Another method for stimulating the MHC class I and II presentation ofepitopes on APC is based on the incorporation of polynucleotides codingfor the desired polypeptides using plasmids, non-viral or viral vectors.A disadvantage of using plasmids for diagnostic purposes is the lowefficiency of the nucleic acid transfer in APC using the hithertoavailable transfection methods, for example electroporation orlipofection. Viral or bacterial vectors frequently have significantlyincreased transfection rates of APCs compared to plasmids. However,these gene transfer systems are frequently not immunologically inert andmodulate the capability of APC for epitope processing and presentationof polypeptides (Jenne et al. (2001), Trends Immunol., 22:102-7). Inaddition, the use of these nucleic-acid-based methods is limited by theexpensive and costly production of gene ferries. Furthermore, so farthere are no examples of application relating to the suitability ofthese systems for the diagnostics of other immune cell populations, forexample, CD4⁺CD8^(dim) cytotoxic T cells, CD56⁺CD8⁺ NKT andCD56⁻CD57⁺CD8⁺ NKT cells.

The detection of the individual populations used after variouspopulations of immune cells have been stimulated will be brieflydescribed here. So far CD4⁺ T helper cells have been detected bydetermining the cell proliferation or the messenger substances(cytokines) produced by T cells after a specific stimulation. The cellproliferation is usually detected using a proliferation assay bydetermining the radioactive isotope ³H tritium incorporated in the DNAof proliferating cells. The cytokine production from CD4⁺ T cells aftera polypeptide-specific stimulation can be determined by means of acytokine ELISA, an ELISPOT assay or by means of FACS technology bydetermining intracellular cytokines or secreted cytokines (FACSsecretion assay).

Polypeptide-specific CD4⁻CD8⁺ cytotoxic T cells (CTL) haveconventionally been detected by detecting their specific cytotoxicactivity or the messenger substances (cytokines) produced by CTL after aspecific stimulation, especially of interferon-γ (IFN-γ). Thecytotoxicity is usually detected by means of a classical chromiumrelease test or adequate non-radioactive method in which the release ofenzymes or ATP from target cells as a result of a specific lysis by theeffector cell with cytotoxic properties is measured.

The cytokine production from CD8⁺ T cells after an epitope-specificstimulation can be determined by means of a cytokine ELISA, an ELISPOTassay or by using FACS technology by determining intracellular cytokinesor secreted cytokines (FACS secretion assay). IFN-γ is usually used as amarker cytokine for the presence of CTL. So far, autologous APC whichpresent CTL epitopes in conjunction with MHC proteins of class I ontheir surfaces, have been used, for example, to stimulate epitope- orpolypeptide-specific CTL. The induction of an MHC class I mediatedepitope presentation on APC has so far been mediated by incubating thiswith epitope-carrying peptides of suitable length (8 to 11 amino acids),by incubating with lipopolypeptides, particular polypeptides orpolypeptides packed in particular structures, lysates ofpolypeptide-producing cells, apoptotic cells as well as vital but killedpolypeptide-producing micro-organisms, especially recombinant viruses,bacteria or yeasts.

So far, polypeptide-specific CD4⁺CD8^(dim) cytotoxic T cells have beendetected by determining the IFN-γ production after a specificstimulation of cells in whole blood by means of inactivated virusparticles, for example, gp120-depleted HIV-1 antigen (Reimmune™ inincomplete Freund's adjuvant) or purified cytomegalovirus (CMV) lysate(Advanced Biotechnologies, Columbia, Md.) (Suni et al. (2001), Eur. J.Immunol., 31:2512-20).

CD56⁺CD8⁺ and CD56⁻CD57⁺CD8⁺ NKT cells possess a very limited T cellreceptor repertoire, which suggests that this cell population can onlyrecognise a limited number of MHC presented antigens. So far, no methodshave been described for detecting NKT cells in the human system.

Dimer and tetramer technology are methods for detecting epitope-specificCD8⁺ cytotoxic T cells and CD4⁺ T helper cells. However, limitations ofthese methods for widespread use in T cell diagnostics are based on thevery high costs for the manufacture of dimers and tetramers. Inaddition, dimer and tetramer technology has so far only been availablefor a limited repertoire of MHC types, especially for frequent MHC classI proteins, for example, HLA A2. In addition, this technique only allowsthe detection of defined epitope-specific T cells. T cell reactivitiesagainst multiple epitopes can only be determined using this method witha substantial expenditure of time and money.

The stimulation of peripheral blood cells with inactivated virusparticles is so far the only method described for the stimulation anddetection of CD4⁺CD8^(dim) cytotoxic T cells.

These stimulants, however, contain a complex mixture of different viraland non-viral polypeptides as well as other partly immune-stimulatoryvirus components such as nucleic acids, lipids and sugars. It is thusnot possible to precisely allocate the T cell reactivity determined todefined viral polypeptides.

It is thus the object of the present invention to provide a detectionsystem with which polypeptide-specific or epitope-specific T cell andother immune cell populations can be detected simultaneously. It isfurther the object of the present invention to provide a new method forthe infiltration of polypeptides into cells, especially APCs.

It is further the object of the present invention to provide a newmethod for inducing the MHC class I and II presentation ofpolypeptide-specific epitopes by means of APCs.

This object is solved by the subject matter defined in the claims.

The following figures are used to explain the invention.

FIG. 1 is a graphical representation showing the number of IFN-γsecreting T cells after incubating peripheral blood mononuclear cells(PBMC) with urea-adjuvated BZLF1 polypeptide. In each case 2×10⁵ PBMC orCD8⁺ T-cell-depleted PBMC from respectively 2 HLA B8-negative,EBV-positive donors (LD, FN), one HLA B8-negative, EBV-negative donor(BH), one HLA B8 positive, EBV negative donor (JW), and 4 HLAB8-positive, EBV-positive donors (JU, SD, MB, RE) were sown in T cellmedium in ELISPOT plates and incubated with 5 μg/ml urea-adjuvated BZLF1polypeptide in each case for 24 hours. The number of IFN-γ secretingcells was then determined using ELISPOT. The values shown aremeans±standard deviation (SD) from 6 independent experiments.

FIGS. 2 a and b show the result of a flow-cytometric analysis of wholeblood in a dotplot representation. An FSC/SSC (Forward Scatter/SideScatter) dotplot is shown wherein the measurement region given by R1(Region 1) corresponds to the lymphocyte population. In the followingfigures only the lymphocytes contained in G1 are shown. G1 correspondsto the population which was determined when restricting the measurementto one window in accordance with the region R1 in FIG. 2.

FIGS. 3 a and b show dotplots giving the frequency and distribution ofvarious populations of CD3 and CD8 positive lymphocytes in whole bloodafter stimulating with urea-adjuvated BZLF1 polypeptide (FIG. 3 a) or asynthetic BZLF1 peptide (RAKFKQLL; Bogedain et al. (1995); J. Virol.69:4872) (FIG. 3B), which contains a known CTL epitope.

Whole blood from an HLA B8-positive Epstein Barr Virus (EBV)-positivedonor was either treated with urea-adjuvated BZLF1 protein or with thesynthetic BZLF1 peptide and stimulated for 6 hours. The activated cellswere then stained with anti-CD3-FITC and anti-CD8-APC. Two distinctpopulations of CD3⁺CD8⁺ cells can be detected by this method. Thepopulations showing a strong expression of CD3 and CD8 polypeptides(shown in Region 2 (R2)), involve classical CD8⁺ cytotoxic Tlymphocytes. The cell population shown in Region 3 (R3) which has areduced expression of CD3 and CD8 polypeptides comprises CD56⁺ NKTcells, as confirmed in FIG. 5. In this case, the urea-adjuvated BZLF1polypeptide (FIG. 3 a) and the synthetic BZLF1 peptide (FIG. 3 b)clearly differ in their capacity to stimulate both populations ofCD3⁺CD8⁺ cells.

FIGS. 4 a and b show dotplots giving the frequency and distribution ofvarious populations of CD4⁺ and CD8⁺ lymphocytes in whole blood afterstimulation with urea-adjuvated BZLF1 polypeptide (FIG. 4 a) or asynthetic BZLF1 peptide (FIG. 4B), which contains a known CTL epitope.Whole blood from an EBV-positive donor was treated either withurea-adjuvated BZLF1 protein or the synthetic BZLF1 peptide andstimulated for 6 hours. The activated cells were then stained withanti-CD4-ECD and anti-CD8-APC. A population of CD4 and CD8 doublepositive CD4⁺CD8^(dim) T-lymphocytes can be detected using this method.After re-stimulating the whole blood of an EBV-positive patient withurea-adjuvated BZLF1 polypeptide (FIG. 4 a), a significantly increasednumber of these double-positive CD4⁺CD8^(dim) cells could be detectedcompared with that after stimulating the same whole blood using theBZLF1 peptide (FIG. 4 b).

FIGS. 5 a and b show dotplots giving cell populations which exhibitexpression of the surface markers CD8 and CD56. It can be clearly seenin both figures (FIGS. 5 a and 5 b) that the population of CD8-positivecells, characterised in FIG. 2 by R3, and shown in blue here, expressesCD56 on the cell surface. Thus, the cells shown in the region R3represent the population of the NKT cells.

FIGS. 6 a and b show dotplots giving the result of a flow-cytometricanalysis of cells from whole blood after stimulating with urea-adjuvatedBZLF1 polypeptide (FIG. 6 a) or a synthetic BZLF1 peptide (FIG. 6B).This is a FSC/SSC (Forward Scatter/Side Scatter) dotplot, wherein themeasurement region given by R1 (Region 1) represents the lymphocytepopulation.

FIGS. 7 a and b show histogram plots giving the result of the IFN-γexpression from various populations of the total lymphocytes registeredin R1 after stimulation with urea-adjuvated BZLF1 polypeptide (FIG. 7 a)or BZLF1 peptide (FIG. 7B). M1 and M2 are set as markers here. M1designates the region in which the IFN-γ expression is to be classifiedas positive. All cells localised to the left of the characterised regionM1 were non-specifically stained by non-specific binding of theanti-IFN-γ antibody within the cells. The region characterised by M2shows the IFN-γ expression by the pure population of CD3⁺CD8⁺ cytotoxicT cells. The difference in the IFN-γ values shown in M1 and M2characterises the IFN-γ production by weakly CD8⁺ cells with NK cellproperties. After re-stimulation with urea-adjuvated BZLF1 polypeptide(FIG. 7 a), compared with peptide stimulation, a significantly increasedIFN-γ production from cells of whole blood as well as increasedstimulation of the population of weakly CD8⁺ cells with NK cellproperties can be observed.

FIGS. 8 a and b show dotplots giving the result of the expression of thesurface marker CD8 against the side scatter (granularity of the cells)of cells from whole blood after stimulation with urea-adjuvated BZLF1polypeptide (FIG. 8 a) or the synthetic BZLF1 peptide (FIG. 8 b). Withthis adjustment CD8⁺ cytotoxic T lymphocytes can be shown. These cellsare designated here as the R3 population.

FIGS. 9 a and b show histogram plots giving the result of the IFN-γexpression of the population of CD8⁺ cytotoxic T cells from whole bloodshown in R3 after stimulation with urea-adjuvated BZLF1 polypeptide(FIG. 9 a) or the synthetic ZLF1 peptide (FIG. 9 b). The markers M1 andM2 are set as in FIGS. 7 a and b. This figure shows that urea-adjuvatedBZLF1 protein and the synthetic BZLF1 peptide are equally suitable fordetermining BZLF1-specific cytotoxic T cells. However, CD4⁺ CD8^(dim)and CD8⁺ T cells could only be read out simultaneously after incubatingthe cells with urea-adjuvated BZLF1 polypeptide.

FIGS. 10 a and b show dotplots giving the result of the expression ofthe surface marker CD56 against the side scatter of cells from wholeblood after stimulation with urea-adjuvated BZLF1 polypeptide (FIG. 10a) or the synthetic BZLF1 peptide (FIG. 10 b). CD56⁺ NKT cells can beshown using this method. This population is designated here as R4.

FIGS. 11 a and b show histogram plots giving the result of the IFN-γexpression of the NKT cell population designated as R4 after stimulationwith urea-adjuvated BZLF1 polypeptide (FIG. 11 a) or the synthetic BZLF1peptide (FIG. 11 b). The markers M1 and M2 are set as in FIGS. 7 a andb. This figure confirms the efficient stimulation of weakly CD8 positiveNKT cell populations using urea-adjuvated BZLF1 polypeptide.

FIG. 12 shows an immunoblot giving the result of the reactivity of apolyclonal rabbit serum against recombinant BZLF1 protein. A rabbit wasinjected intramuscularly with 30 μg of urea- and Hunters Titermaxadjuvated BZLF1 polypeptide and re-immunised with the same immunogenafter 4 and 8 weeks. After a further 3 weeks, serum of the animal in adilution of (A) 1:2000, (B) 1:10000 and (C) 1:50000 in the immunoblotwas tested for its reactivity towards recombinant BZLF1-protein inconcentrations of (1) 20 ng, (2) 100 ng and (3) 500 ng.

FIG. 13 is a schematic diagram showing the dose dependence of the IFN-γsecretion after stimulation with urea-treated BZLF-1 protein. Thestudies showed that BPMCs from 2 of the 4 tested HLA-B8-positive,EBV-positive donors at all the tested BZLF-1 concentrations showed asignificantly increased number of IFN-γ producing BZLF-1-specific Tcells compared to the negative controls (donors 1-3). In theseexperiments, low concentrations of urea-adjuvated BZLF-1 protein weresufficient to detect a significantly increased number of BZLF-1specific, IFN-γ producing T cell compared to the negative controls(donors 1-3) in 3 out of 4 HLA-B8-positive, EBV-positive donors.

FIG. 14 is a schematic diagram showing the time behaviour of IFN-γsecretion after stimulation with urea-treated BZLF-1 protein. Thestudies showed that after incubation for 8 hours, all HLA-B8-positive,EBV-positive donors (donors 4-7) showed a significantly increased numberof IFN-γ producing, BZLF-1-specific T cells compared to all the “controlprobands” (donors 1-3). The maximum number of IFN-γ producing cellscould be observed after 16-24 hours depending on the donor.

FIG. 15 is a graph showing the influence of the urea concentration onthe vitality of PBMCs. D.H., S.B. and I.K. designate the donors.

Generally used abbreviations for nucleotides and amino acids are used inthe present invention.

The term “genomic” used here denotes the entirety or fragments of thegenetic material of an organism.

The term “polynucleotide” used here denotes the polymeric form ofnucleotides of arbitrary length, preferably deoxyribonucleotides (DNA)or ribonucleotides (RNA). The term only denotes the primary structure ofthe molecule.

The term includes double- and single-stranded DNA or RNA, e.g. decoy orantisense polynucleotides.

The term “polypeptide” used here denotes a polymer of amino acids ofarbitrary length. The term polypeptide also comprises the terms epitope,peptide, oligopeptide, protein, polyprotein and aggregates ofpolypeptides. Also included in this term are polypeptides which havepost-translational modifications e.g. glycosylations, acetylations,phosphorylations and similar modifications.

This term furthermore comprises, for example, polypeptides which haveone or a plurality of analogs of amino acids (e.g. unnatural aminoacids), polypeptides with substituted links as well as othermodifications which are state of the art, regardless of whether theyoccur naturally or are of non-natural origin.

The term “urea-adjuvated” used here means that a molecule, e.g., apolypeptide is present in a solution, preferably an aqueous solutionsuch as water, a buffer solution, a cell culture medium or a body fluidwhich has a certain urea concentration. The term “urea-adjuvated” thusmeans that the polypeptides are not only denatured by urea but are alsobrought in contact with cells for transfection in a urea-containingsolution. The urea concentration of the denaturing and transfectionsolution can be identical or different. Furthermore, the urea solutioncan contain NaCl and/or DTE. The urea concentration of the transfectionsolution preferably has a final concentration in the range of about0.001 to about 0.8 mol/litre, especially preferably in the range ofabout 0.001 to about 0.2 mol/litre, further from about 0.001 to about0.1 mol/litre, especially preferably from about 0.001 to about 0.01mol/litre, further especially preferably from about 0.01 to about 0.2mol/litre, further from about 0.01 to about 0.1 mol/litre furtherespecially preferably from about 0.1 to about 0.8 mol/litre. However,the urea concentration can also be less than 0.001 mol/litre or morethan 0.8 mol/litre. If there is a high total number of living cells anda ratio of living to dead cells where the living cells predominate, theurea concentration of the transfection solution should be less than 0.3mol/litre, preferably for example. 2.9, 2.8, 2.7, 2.6, 2.5 or anyconcentration less than 0.3 mol/litre.

The terms “purified”, “purified” and “isolated” mean that a molecule,for example, a polypeptide or a nucleic acid sequence is present in thecompletest possible absence of biological macromolecules of comparabletype. The terms mean a fractional weight of the desired product of atleast 75%, preferably of at least 85%, particularly preferably of atleast 95% and especially preferably of at least 98% to the total weightof the biological macromolecules present (water, buffer and other smallmolecules, especially molecules having a molecular mass of less than1000 are not included with the biological macromolecules).

The term “epitope” used here designates the region of a polypeptidewhich possesses antigen properties and for example serves as arecognition site of T cells or immunoglobulins. In the sense of thisinvention epitopes for example are those regions of polypeptides whichare recognised by immune cells such as, for example, CD4⁺ T helpercells, CD8⁺ cytotoxic T cells, CD4⁺CD8^(dim) cytotoxic T cells,CD56⁺CD8⁺ and CD56-CD57⁺CD8⁺ NKT cells or CD4⁺CD25⁺ T suppressor cells.An epitope can comprise 3 or more amino acids. Usually, an epitopeconsists of at least 5 to 7 amino acids or, which is more common, 8-11amino acids, or however of more than 11 amino acids, or however of morethan 20 amino acids, even more rarely of more than 30 amino acids. Theterm “epitope” also comprises a unique spatial conformation for theepitope. This spatial conformation is obtained from the sequence ofamino acids in the region of the epitope.

The term “micro-organism” used here designates viruses, prokaryotic andeukaryotic microbes, such as for example archaebacteria, single cellsand fungi; the latter group for example comprises yeast and filamentousfungi.

The term “immune cells” used here denotes lymphocytes with regulatory orcytolytic properties such as, for example, CD4⁺ T helper cells, CD8cytotoxic T cells, CD4⁺CD8^(dim) cytotoxic T cells, CD4⁺CD25⁺ Tsuppressor cells, CD56⁺CD8⁺ and CD56⁻CD57⁺CD8⁺ NKT cells as well as NKcells.

The term “antigen-presenting cell” (APC) used comprises cells which arecapable of capturing, processing and presenting fragments of thesepolypeptides (epitopes) to the immune system in association with MHCclass I and MHC Class II proteins. In particular, the term“antigen-presenting cell” comprises dendritic cells (Langerhans cells),monocytes, macrophages, B cells but also vascular epithelial cells andvarious epithelial, mesenchymal cells as well as microglia cells of thebrain.

The present invention relates to a method for polypeptide transfer incells, comprising the following steps:

-   -   a) Incubating polypeptides with a urea solution,    -   b) Incubating cells with the polypeptides present in the urea        solution.

The polypeptides can be synthetically produced polypeptides or they canbe expressed in various cells by means of usual pro- or eukaryoticexpression systems, wherein the following list only contains a few cellsas examples, e.g. bacteria such as Bacillus subtilis, E. coli,Streptococcus cremoris or Streptococcus lividans, yeast cells such asCandida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromycesfragilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, orYarrowia lipolytica, insect cells such as Aedes aegypti, Autographacalifornica, Bombvx mori, Drosophila melanogaster, Spodopterafrugiperda, or Trichoplusia ni, mammalian cells such as Chinese hamsterovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkeykidney cells (COS), or plant cells. Said systems for expression ofpolypeptides have been published many times and are state of the art.

The polypeptides used can be purified by conventional molecularbiological methods, e.g. by cell disruption, nucleic acid digestion,methods for concentrating proteins, affinity chromatography, ionexchanger chromatography and gel filtration, or in combination withpolypeptide-denaturing methods e.g. acid precipitation, urea treatment,alkali treatment, heat treatment, treatment with sodium dodecylsulphate(SDS) or sonification. These methods for purifying polypeptides canfurthermore be combined in an arbitrary fashion.

The peptides can be synthetic i.e. non-naturally occurring polypeptidesor they can occur in arbitrary living beings, e.g. in mammals such ashumans, primates, mouse, rat, rabbit, sheep, cow, pig or in any animals,parasites, micro-organisms or viruses. However, they can also originatefrom plants and algae. In addition, they can originate from prionproteins.

The concentration of urea solution in step a) is preferably in the rangeof about 0.01 mol/litre to about 10 mol/litre or even higher, preferablyup to about 0.1 mol/litre, particularly preferably in the range of about0.1 to about 0.5 mol/litre, especially preferably in the range of about0.5 to about 5 mol/litre, further especially preferably from about 5 toabout 1.0 mol/litre or is more than 10 mol/litre.

The polypeptides can be used in any arbitrary concentrations, theconcentration preferably lies in the range of about 0.01 to about 50μg/μl or higher, preferably in the range of about 0.01 to about 0.1μg/μl, particularly preferably from about 0.1 to about 0.5 μg/μl,especially preferably from about 0.5 to about 2 μg/μl, furtherespecially preferably from about 2 to about 10 μg/μl or from about 10 toabout 50 μg/μl or more than 50 μg/μl.

The incubation time of the polypeptides with the urea solution can beless than 10 seconds, or about 10 seconds up to one day, preferablyabout one minute, or about one minute up to about one hour, particularlypreferably about one hour or more than one day. The polypeptides canfurthermore either be stored frozen or at a temperature at which theurea solution is in the liquid state, for any arbitrarily long time inthe urea solution, e.g., for more than one week, for more than one monthor for more than one year.

The polypeptides present in the urea solution are then used forincubating the cells. Such a large volume of the polypeptide/ureasolution to the cells is used that the concentration of polypeptides forapproximately 10⁶ cells is in the range of about 0.1 to about 200 μg orhigher, preferably in the range of about 0.1 to about 200 μg,particularly preferably from about 0.1 to about 2 μg, especiallypreferably from about 0.1 to about 10 μg, further especially preferablyfrom about 10 to about 50 μg or from about 50 to about 200 μg ofpolypeptide.

The urea concentration in step b) should preferably have a finalconcentration in the range of about 0.001 to about 0.8 mol/litre,particularly preferably in the range of about 0.001 to about 0.2mol/litre, further from about 0.001 to about 0.1 mol/litre, especiallypreferably from about 0.001 to about 0.01 mol/litre, further especiallypreferably from about 0.01 to about 0.2 mol/litre, further from about0.01 to about 0.1 mol/litre, further especially preferably from about0.1 to about 0.8 mol/litre. However the urea concentration can also beless than 0.001 mol/litre, or more than 0.8 mol/litre. If there is ahigh total number of living cells and a ratio of living to dead cellswhere the living cells predominate, the urea concentration in step b)should be less than 0.3 mol/litre, preferably, for example, 2.9, 2.8,2.7, 2.6, 2.5 or any concentration less than 0.3 mol/litre. In addition,the urea solution can contain NaCl and/or DTE, wherein the concentrationof NaCl is in the range of about 0.25 mmol/litre to about 0.2 mol/litreand that of DTE is in the range of about 0.25 nmol/litre to about 0.2mmol/litre. Furthermore, the DTE concentration can be less than 0.25nmol/litre or greater than 0.2 mmol/litre.

The polypeptide/urea solution is incubated with the cells for about 1 toabout 240 hours or longer, preferably for about 2 to about 6 hours orfor about 6 to about 12 hours or for about 12 to about 36 hours, or forabout 36 to about 240 hours.

The method according to the invention can be used to infiltratepolypeptides into arbitrary cells, that is prokaryotic e.g. bacteria andeukaryotic cells, e.g. fungi such as yeasts and filamentous fungi,insect cells, bird, reptile, fish, amphibian, mammalian cells e.g.,murine or human cells e.g. antigen-presenting cells.

In contrast to the methods described hitherto for polypeptide transferin cells, the method according to the invention is especiallydistinguished by the fact that it is universally applicable, simple toimplement with high efficiency and the cost is very low.

The cells treated using the method according to the invention can beused in the area of research, diagnostics and treatment and preventionof diseases in animals and humans. For example, the APCs obtained usingthe method according to the invention are suitable for prophylactic andtherapeutic applications for combating infectious diseases and tumours.Furthermore, the APCs obtained using the method according to theinvention are suitable for the simultaneous diagnostics of a wide rangeof polypeptide-specific and immunogen-stimulatable immune cells,comprising CD4⁺CD8⁻ T-helper cells, CD4⁻CD8⁺ cytotoxic T cells,CD4⁺CD8^(dim) cytotoxic T cells, CD4 CD25 T-suppressor cells, CD56⁺CD8⁺and CD56⁻CD57⁺CD8⁺ NKT-cells or CD56⁺ NK cells. Thus, the inventionfurther relates to cells obtained using the method according to theinvention and their use.

A further aspect of the present invention relates to the use ofurea-adjuvated polypeptides in a plurality of different scientific,medicinal and diagnostic applications, e.g. for studying the importanceof these polypeptides in cellular processes, for inducing humoral andcellular immune responses in experimental animals and in humans, for (a)obtaining sera and antibodies for diagnostic, therapeutic and preventiveapplications, (b) for inducing suitable immune responses to protectagainst or for the treatment of microbial infections and tumourdiseases, as (c) prophylactic and therapeutic vaccines or (d) for the exvivo stimulation of APC for diagnostic, therapeutic and preventivepurposes.

Thus, the present invention relates to the use of urea-adjuvatedpolypeptides for inducing specific antibodies and T-cells in animals,especially in mammals, e.g., in mice, rats, rabbits, sheep, horses,cattle, pigs, dogs, cats and primates. The urea-adjuvated polypeptidescan also be used for inducing humoral and cellular immune responses inhumans. In this case, the urea-adjuvated polypeptides can either beadministered alone or in combination with immune-stimulating agents(“adjuvants”).

Especially suitable adjuvants for enhancing the efficiency of thevaccines/vaccine combinations described include, for example: (1)gel-like adjuvants such as aluminium salts (Alum), such as aluminiumhydroxide, aluminium phosphate, aluminium sulphate and calciumphosphate; (2) microbial adjuvants such as bacterial nucleic acids withCpG motifs, endotoxins such as, for example, monophosphoryl lipid A,exotoxins such as for example, the diphtheria, cholera, tetanus toxoid,the heat-labile enterotoxin of E. coli and muramyl dipeptides such as,for example, MDP; (3) oil emulsions and emulsion-based vaccines such as,for example, incomplete Freund's adjuvant (IFA), MF59, SAF and Ribi™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.); (4)particular adjuvants such as, for example, immune-stimulatory complexes(ISCOMs), liposomes, PLG polymers, biologically degradable microspheresand saponins (QS-21), and synthetic adjuvants such as non-ionic blockpolymers, muramyl peptide analogues, polyphosphazene and syntheticpolynucleotides and (5) cytokines, such as for example interleukins(IL-1, IL-2, IL-12 among others.), granulocyte/macrophagecolony-stimulating factor (GM-CSF) or macrophage colony stimulatingfactor (M-CSF), as well as the tumour necrosis factor (TNF). In additionto adjuvants, all other substances which have an immune-stimulatingeffect to enhance the efficiency of the vaccine compositions describedcan be used. A listing of suitable available adjuvants has beencompiled, for example, by F. R. Vogel and can be retrieved via the worldwide web at the following address(hppt://www.niaid.nih.gov/aidsvaccine/pdf/compendium.pdf).

The vaccine combinations described (e.g., urea-adjuvated polypeptides inconjunction with a pharmaceutically accepted carrier component and/or anadjuvant) are added to the dilution solutions, such as for example,water, salt solutions, glycerol, ethanol. In addition, additionalaccessory components such as moistening and emulsifying agents,pH-buffering substances and similar components can be present in thesecompositions.

These vaccine combinations are usually present in injectable form,either as liquid solutions or suspensions.

The immunogen composition can equally be emulsified or incorporated inliposomes in order to achieve enhanced adjuvant properties in the senseof a pharmaceutically accepted carrier component. The vaccinecomposition can be administered by suitable administration routes.Possible among others in this case are oral, topical, intravenous,intraperitoneal, intramuscular, intra-articular, subcutaneous,intranasal or intradermal administration routes. The vaccine compositionis used in the appropriate dosage for the indication. The determinationof an appropriate dosage for various organisms is state of the art. Thevaccine combinations described can either be used prophylactically ortherapeutically. Furthermore, APC modified using urea-adjuvatedpolypeptides are suitable for therapeutic and preventive applications.Methods for obtaining and ex vivo expansion of APC, as well as forreinfusion of ex vivo modified APC in an organism have been published onmany occasions and are state of the art.

A further aspect of the present invention relates to a method fordetecting polypeptide-specific immune cells, comprising the followingsteps:

-   -   a) Incubating polypeptides with a urea solution,    -   b) Incubating APC-containing cell cultures or body fluids with        the polypeptides present in the urea solution,    -   c) Incubating the APC-containing cell cultures or body fluids        obtained according to step b) with immune cells or        immune-cell-containing body fluids,    -   d) Simultaneously detecting and/or quantifying various subtypes        of immune cells which are specific against the polypeptides from        step a).

Step a) of the method according to the invention is identical to step a)of the method for polypeptide transfer in cells so that thecorresponding preceding explanations also apply to the method fordetecting polypeptide-specific immune cells.

The polypeptides present in the urea solution are then used forincubating the APC-containing cell cultures or body fluids. Such a largevolume of the polypeptide/urea solution to the cells is used that forapproximately 10⁶ cells the concentration of polypeptides is in therange of about 0.1 to about 200 μg or higher, preferably in the range ofabout 0.1 to about 200 μg, particularly preferably from about 0.1 toabout 2 μg, especially preferably from about 2 to about 10 μg, furtherespecially preferably from about 10 to about 50 μg, or from about 50 toabout 200 μg of polypeptide.

The urea concentration in step b) should preferably have a finalconcentration in the range of about 0.001 to about 0.8 mol/litre,particularly preferably in the range of about 0.001 to about 0.2mol/litre, further from about 0.001 to about 0.1 mol/litre, especiallypreferably from about 0.001 to about 0.01 mol/litre, further especiallypreferably from about 0.01 to about 0.2 mol/litre, further from about0.01 to about 0.1 mol/litre, further especially preferably from about0.1 to about 0.8 mol/litre. However the urea concentration can also beless than 0.001 mol/litre, or more than 0.8 mol/litre. If there is ahigh total number of living cells and a ratio of living to dead cellswhere the living cells predominate, the urea concentration in step b)should be less than 0.3 mol/litre, preferably, for example, 2.9, 2.8,2.7, 2.6, 2.5 or any concentration less than 0.3 mol/litre. In addition,the urea solution can contain NaCl and/or DTE, wherein the concentrationof NaCl is in the range of about 0.25 mmol/litre to about 0.2 mol/litreand that of DTE is in the range of about 0.25 nmol/litre to about 0.2mmol/litre.

Furthermore, the DTE concentration can be less than 0.25 nmol/litre orgreater than 0.2 mmol/litre.

The polypeptide/urea solution is incubated with the APC-containing cellcultures or body fluids for about 1 to about 240 hours or longer,preferably for about 2 to about 6 hours or for about 6 to about 12 hoursor for about 12 to about 36 hours, or for about 36 to about 240 hours

The APC-containing cell cultures can be purified PBMC population(leukapheresate), isolated monocytic cells or a separated APCpopulation, e.g., dendritic cells (Langerhans cells), monocytes,macrophages or B cells. The term “APC-containing cell cultures” as usedhere thus means not only cells comprising APC held and multiplied invitro in culture media but also cell populations taken from a proband,patients or an animal and containing purified APC.

The APC-containing body fluid is preferably whole blood or liquor.

For example, blood or another APC-containing body fluid can be takenfrom a proband or patient. The body fluid can either be used directly instep b) of the method according to the invention or APC-containing cellpopulations can be purified and then used. The purification ofAPC-containing cell populations from blood or other APC-containing bodyfluids is state of the art and known to the person skilled in the art.

After incubating the APC-containing cell cultures or body fluids withthe polypeptides present in the urea solution, the cells are incubatedwith immune cells or immune-cell-containing body fluids. The immunecells or immune-cell-containing body fluids preferably come from thesame proband, patient or animal from which the APC-containing cellcultures or body fluids originate. Alternatively, the immune cells orimmune-cell-containing body fluids also come from probands, patients oranimals having an MHC pattern compatible with the APC-containing cellcultures or body fluids.

The immune cells can be T cells, e.g., CD4⁺ T cells, CD8⁺ T cells,CD4⁺CD8^(dim) T cells, CD4⁺CD25⁺ suppressor T cells, but also other cellpopulations such as, for example, CD56⁺CD8⁺ and CD56⁻CD57⁺CD8⁺ NKT cellsor CD56⁺NK cells. Furthermore, the term immune cells also comprises anarbitrary mixture of CD4⁺ T cells, CD8⁺ T cells, CD4⁺CD8^(dim) T cells,CD4⁺ CD25⁺ T cells, CD56⁺CD8⁺ as well as CD56⁻CD57⁺CD8⁺ NKT cells andCD56⁺ NK cells. The term “immune cells” as used here thus means not onlyimmune cells held and multiplied in vitro in culture media but alsoimmune cell populations taken from a proband, patient or an animal andpurified. The immune-cell-containing body fluids are preferably wholeblood or liquor. Methods for obtaining and purifying defined APC andimmune cell populations have been published on many occasions and arestate of the art.

The APC-containing cell culture or body fluid, e.g. whole blood, liquoror purified PBMC used in step b) can already contain the populations ofimmune cells to be detected. In this case, it is no longer necessary toadd immune cells or immune-cell-containing body fluids in step c).

The incubation in step c) is about 1 to about 240 hours or longer,preferably about 2 to about 6 hours or about 6 to about 12 hours orabout 12 to about 36 hours, or about 36 to about 240 hours undersuitable cultivation conditions, for example, at 37° C. in a humidifiedatmosphere with 5 to 8% CO₂ in T cell medium (RPMI 1640 with 2 to 10%heat-inactivated (30 min, 56° C.) human serum or foetal calf serum(FCS), 2 mM glutamine and 100 mg/ml kanamycin or gentamicin (allcomponents from PanSystems, Aidenbach)).

Other suitable conditions (variation of the media composition,temperature, air humidity, incubation time) for cultivating APC andimmune cells have been described on many occasions and are state of theart.

The detection of defined populations of polypeptide-specific immunecells is based on the finding that after a specific recognition ofpolypeptides which are presented jointly with MHC proteins of classes IIand/or I on the surface of APC, immune cells show an enhanced expressionof characteristic cytokines, especially IFN-γ, or IL4 and/or IL5. As aresult of a joint analysis of surface proteins which are characteristicof defined immune cell populations, and of cytokines, the presenceand/or concentration of defined populations of polypeptide-specificimmune cells can be detected from a mixture of different populations ofimmune cells. The detection and/or quantification in step d) thus takesplace via the simultaneous detection of surface proteins and cytokines.

The detection of defined cell populations via specific surface proteinsis carried out, for example via CD4 for T helper cells, CD8 forcytotoxic T cells, CD4 and CD8 for CD4⁺ CD8^(dim) cytotoxic T cells,CD56 for NK cells, CD4 and CD25 for suppressor T cells, and CD56 and CD8or CD57 and CD8 for various populations of NKT cells. Specific states ofthe cell populations (inactive versus activated cells versus memorycells) and the degree of activatability can furthermore be determined bydetecting additional surface proteins (for example, CD69, CD45RA, CCR7)and intracellular proteins (for example, granzyme or perforin).

These characteristic surface markers for defined cell populations havebeen published on many occasions and the detection and characterisationof different populations of immune cells using FACS technology forexample is state of the art.

The specific activation of immune cells is detected after incubatingwith the APC-containing cell cultures or body fluids obtained accordingto step b) by measuring any increased cytokine production of theactivated immune cells. In this case, for example, CD4⁺ T helper cellsof the T-helper 1 type (Th-1), CD8⁺ cytotoxic T cells, CD4⁺CD8^(dim)cytotoxic T cells, CD56⁺ NK cells, and CD56⁺CD8⁺ or CD57⁺CD8⁺ NKT cellsafter specific stimulation produce increased IFNγ, whereas CD4⁺ T helpercells of the T helper 2 type (Th-2) show increased production of thecytokines IL4 and IL5. The cytokines produced can be determined simplyusing methods published on many occasions either intracellularly orafter secretion in the supernatant using, in some cases, commerciallyavailable methods, for example, using FACS technology. Detection is alsopossible by means of other cytokines produced after the specificactivation of immune cells or other markers produced.

Reactive immune cells can be determined and characterised for exampleusing Fluorescence activated cell scan (FACS) technology. This methodallows the fluorescence intensity of individual cells in a mixed cellpopulation to be measured using a flow cytometer. The flow-cytometricanalysis of the cells is then made using an FACS system for example anFACS CALIBUR, Becton Dickinson (Franklin Lakes, N.J., USA), and theevaluation is made using the Cell Quest program (Becton Dickinson,Heidelberg).

Fluorescence-coupled, e.g. with R-phycoerythrin (R-PE),peridin-chlorophyll c (PerCP), fluoescein (FITC), Texas Red (TX),allophycocyanin (APC), Tandem PE-TX, Tandem PE-Cy5, PE-Cy7 or TandemAPC-Cy7, primary or secondary antibodies are suitable for detecting thecharacteristic surface proteins and cytokines described previously usingFACS technology and are available commercially (for example, from BectonDickinson, Dako, Coulter). In addition to the FACS method, other methodssuitable for determining the production from immune cells, for exampleELISA methods, Elispot methods and biosensors, are also suitable fordetecting polypeptide specific immune cells. These methods fordetermining cytokines have been described on many occasions and arestate of the art.

The method for detecting polypeptide-specific T cells according to theinvention is suitable for many different scientific and medicalapplications, e.g. in analysis, diagnosis and therapy. Using the methodaccording to the invention, different populations ofpolypeptide-specific immune cells can be identified at the same time,for example, for monitoring the efficiency of therapeutic andprophylactic vaccinations for inducing immune cells, for determining theefficiency of therapeutic treatments of diseases involving immune cells,for screening the safety and efficiency of medicaments which causedeletion of anergy of immune cells or bring about a general immunesuppression, for monitoring and diagnosis of diseases induced bymicro-organisms and parasites involving immune cells, for monitoring anddiagnosis of chronic inflammations involving immune cells, formonitoring and diagnosis of tumour-antigen-specific immune cells, formonitoring and diagnosis of immune cells which play a role in transplantrejection, for the diagnosis of autoimmune diseases or for the specificselection of probands for vaccine trials and the testing of therapeutictreatments.

The method according to the invention is used in all vertebrates whichhave immune cells, especially T cells, especially in humans, primatesand rodents. Polypeptide-specific immune cells can be detected andquantified for example from patients suffering from a microbialinfection, a tumour disease, a chronically inflammatory disease, atransplant rejection or an autoimmune disease or however from healthyprobands or participants of therapeutic or preventive trials. Inaddition, epitope-specific T cells can be detected using cells obtainedby the method according to the invention, preferably using APCs obtainedin primates or other animals which possess epitope-specific immunecells.

In contrast to the methods for the detection of polypeptide-specificimmune cells described so far, the method according to the invention ischaracterised by the following advantages:

-   -   (1) Different populations of polypeptide-specific immune cells        can be detected at the same time.    -   (2) The quantity and quality of different populations of        polypeptide-specific immune cells can be detected at the same        time.    -   (3) The method can easily be carried out using commercially        available equipment (FACS) routinely used in many diagnostic        laboratories.    -   (4) The method can be universally used to detect reactive        polypeptide-specific immune cells regardless of the haplotype of        the proband/patient.    -   (5) The diagnostic method according to the invention is suitable        for the patient-specific determination of lymphocyte reactivity        against a complex polypeptide. In this case, it is not necessary        to know target epitopes of these immune cells to carry out the        method.    -   (6) Compared to the conventional diagnostic methods (CT assay,        ELISPOT, cytokine ELISA, proliferation assay), the method of        detection for polypeptide-specific immune cells can be used        universally, is easier to handle, significantly cheaper, less        time-consuming and more sensitive.

The invention is explained using the following examples but is notrestricted to these:

EXAMPLE 1 Production and Purification of the Epstein Barr Virus (EBV)BZLF1 Polypeptide in E. coli

The cDNA for the BZLF1 protein of the EBV strain B95-8 was amplifiedfrom the pCMVZ plasmid (Manet et al. (1990); EMBO 8:1819) by means ofPCR using suitable PCR conditions (introductory denaturing step: 94° C.,2 min; followed by a three-stage PCR (15 cycles) with the followingconditions: denaturing: 94° C., 30 sec; annealing 52° C., 1 min.;elongation: 72° C., 2 min; then again (25 cycles) with the followingconditions: denaturing: 94° C., 30 sec; annealing 62° C., 1 min.;elongation: 72° C., 2 min; and a concluding polymerisation step at 72°C. for 10 min. followed by continuous cooling at 4° C.). In the PCRreaction to amplify the BZLF1 cDNA, 2.5% DMSO was added to the PCRformulation.

The primer A (5′ primer: 5′-GGCGGAGATCTTTAGAAATTTAAGAGATCC-3′; SEQ IDNO:1) and primer B (3′ primer: 5′-GGCGGGGAATTCATGATGGACCCAAACTCG-3′; SEQID NO:2) were used for the amplification. The band amplified by means ofthe PCR was then cleaved with the restriction enzymes BglII and EcoRIand the band obtained in this fashion was then ligated into theplasmid-pET (New England Biolabs) also linearised with BglII and EcoRI.The vector thus produced was called pET5c-Z. The bacterial expressionvector pET5c-Z was then transformed into the E. coli strainBL21-CodonPlus (DE3)-RIL (Carstens and Waesche (1999); Strategies;12:49) and the transformed bacteria were cultured for 1 hour at 37° C.on LB Medium. Thereafter 50 μl of the culture was smeared on LB_(AMP)plates and this was cultured overnight at 37° C. in an incubator. Asingle colony was then inoculated in 200 ml of LB Medium and this wasincubated overnight at 37° C. Then, 10×1 litre of LB Medium wasinoculated in each case with 1/50 volume of the total volume from thepre-culture and the cultures were then cultivated at 26° C. until anO.D.₆₀₀ value of 0.8 was reached. The cultures were then mixed with 1 mMIPTG to stimulate the protein production and cultured further overnight.

To harvest the cells the culture was centrifuged for 10 min. at 5000 rpmin a GS 3 Rotor in a Sorvall cool centrifuge and the cell pelletobtained was resuspended in 250 ml of disintegration buffer (50 mM TrisCl pH 8.0+0.3M NaCl+1 mM EDTA). The resuspended pellet was then frozenaway at −20° C. for further processing. After thawing the cell pelletagain, a spatula tip of lysozyme (muraminidase) was added to the cellsuspension for the cell lysis and the formulation was incubated for 20min at room temperature.

Then, 2 mM of the protease inhibitor Pefablock® was added to theformulation and this was then subjected to an ultrasound treatment onice (3×1 min. at stage 6, pulsation: 80% using a Branson Sonifier,standard tip).

Then, 5 mM MgCl₂ and 2 U Benzonase®/ml protein extract was added to theformulation, the formulation was incubated for 20 min at 37° C. and then(sound suspension), centrifuged in a GSA-Rotor for 30 min. at 14000 rpmand 4° C. and the supernatant discarded. The pellet was then subjectedto a fractionating washing with urea. In this case, after ultrasounddisintegration the pellet was resuspended in 250 ml of 1M urea (in PBSwithout bivalent ions with 2 mM DTE) and the suspension was incubatedfor 1 hour in an overhead shaker at 4° C. The suspension was thencentrifuged for 20 min. at 14000 rpm and the supernatant discarded. Thepellet was then dissolved a second time, as described previously, byvortices in 1M urea, the suspension was centrifuged for 20 min. at 14000rpm and the supernatant discarded. The pellet was then resuspended in250 ml of 2 M urea (in PBS without bivalent ions with 2 mM DTE) and thesuspension was incubated overnight in an overhead shaker at 4° C. Thesuspension was then centrifuged for 20 min. at 14000 rpm, the pellet wasdiscarded and the supernatant was subjected to an acid precipitation.For this purpose the supernatant was titrated with 1N HCl to pH 3.5 andthen centrifuged for 10 min at 12000 rpm. The pellet was discarded andthe supernatant was dialysed 3× against a large volume (3.35 litre) ofdialysis buffer (4M urea, 20 mM Tris Cl pH 7.5, 2 mM DTE). Thesuspension was then applied to a Poros QE anionic exchanger column andthe column was washed with 15 column volumes of running buffer (4M urea,20 mM Tris Cl pH 7.5). The protein was then eluted with 10 columnvolumes of 8M urea, 2M NaCl and various fractions were collected. Thecontent and the purity of the BZLF1 protein in different fractions wasdetermined by means of Coomassie and silver staining of the polypeptidesseparated by SDS-PAGE and by immuno blotting. Fractions which exhibitthe BZLF1 protein with high purity were combined and mixed with 2 mMDTE. The BZLF1-containing suspension obtained was concentrated to about5 ml using an Amikon stirring cell and a YM10 filter (disintegrationvolume 10 kDa) and the proteins in suspension were further separated bygel filtration chromatography. A Superdex 200 prep grade,Pharmacia-HiLoad, 16/60. 120 ml CV, flow rate: 0.5 ml/min was used forthis purpose. The elution of the BZLF1 protein took place after about0.4 column volumes. The clean BZLF1 fractions were combined and theprotein content determined.

With this method the BZLF1 protein can be obtained with a yield of 3.9mg/1 litre of culture and a purity of >95%.

After the purification the BZLF1 protein is present with a concentrationof 0.2 to 1 mg/ml in 8 M urea, 2 mM DTE.

EXAMPLE 2 Detection of BZLF1-Specific Cytotoxic T Cells (CTL) fromPeripheral Blood of an EBV Positive Donor

Testing of the suitability of urea-denatured polypeptide and polypeptidepresent in urea solution for inducing an epitope presentation on MHCclass I and II proteins as well as for simultaneously detectingpolypeptide-specific CD4⁺ and CD8⁺ T cells in mixed APC/lymphocytecultures was carried out using a very well characterised model system.

This is based on the observation that all HLA B8-positive, EBV-positiveprobands possess CD8⁺ cytotoxic T cells which recognise a specificepitope (RAKFKQLL; amino acid 190-197; SEQ ID NO:3) within the EBVprotein BZLF1 (Bogedain et al. (1995); J. Virol. 69:4872; Pepperl etal., (1998) J. Virol. 72:8644). In order to carry out the stimulationexperiments, peripheral blood lymphocytes (PBMC) were obtained by meansof density gradient centrifugation from heparinised whole blood (heparinfinal concentration: 25 IU/ml) of various EBV-negative, HLA B8-positive,or EBV-positive, HLA B8-negative or EBV-positive, HLA B8-positiveprobands. For this purpose 15 ml of Ficoll (PAN, Aidenbach) was placedin special 50 ml Leucosep tubes (Falcon, Becton Dickinson, Heidelberg)and coated with whole blood (diluted 2:1 with PBS). During thesubsequent centrifugation (30 min, 800×g, swing-out rotor, roomtemperature), separation took place into plasma, lymphocyte populationand erythrocytes. The desired lymphocyte population was removed andwashed twice in approximately 30 ml of PBS. In each case, the cells weresedimented by brief centrifugation (5 min, 250×g, room temperature). Thecell pellet was then taken up into T cell medium, the cell number wasdetermined and the cells were either used in the correspondingexperiments.

The depletion of CD8⁺ cells from freshly prepared PBMC took place bynegative selection using CD8 immunomagnetic MicroBeats following themanufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach). Freshlyisolated PBMC was washed, centrifuged (300×g, 10 min, 4° C.) and thecell pellet was resuspended in 80 μl PBS/10⁷ PBMC. The desired cellpopulation was magnetically marked by adding 20 μl of the specificMicrobeads/10⁷ PBMC during an incubation of 15 min at 4° C. The cellsuspension was then washed twice (15 ml PBS), centrifuged and taken upin 500 μl PBS. For the magnetic separation MS⁺ columns (for a maximum of1×10⁷ marked cells) or LS⁺ columns (for a maximum of 1×10⁸ marked cells)were used according to the cell number, equilibrated with 5 ml PBS, thecell suspension was applied and washed with 3×500 μl (MS⁺ columns) or3×5 ml (LS⁺ columns) PBS. Non-marked cells pass through the column. Thecolumn was then removed from the magnetic field and the marked cellpopulation was eluted by adding 500 μl (MS⁺ columns) or 5 ml (LS⁺columns).

BZLF1-specific CD4⁺ T helper cells and CD8⁺ CTL were detected by meansof an enzyme-linked immunospot assay (Elispot). Using Elispot,antigen-specific T cells can be detected using their cytokine secretion(especially the secretion of IFN-γ). Nitrocellulose-coated 96-wellmicrotitre plates (MAHA S 45, Millipore, Eschborn) were coated withmonoclonal, anti-human IFN-□ (Hölzl, Cologne) (5 μg/ml in PBS) andincubated overnight at 4° C. The antibody solution was pipetted off andthe plates were washed four times with 200 μl PBS/formulation in eachcase.

In a following step non-specific binding sites were blocked by adding200 μl of blocking buffer in each case (T cell medium with 10% FCS)during a one-hour incubation at 37° C. PBMC was used in a concentrationof 2×10⁵ cells/formulation in a volume of 100 μl of T cellmedium/formulation (5 replicates each). Stimulation took place directlyin the plate by adding 50 μl of antigen solution (final concentration:BZLF1: 5 μg/ml). After incubation for 24 hours (37° C., 5% CO₂) the cellsuspension was drawn off and any cells still adhering were removed bywashing five times (200 μl of PBS with 0.05% Tween 20 in each case, 5min incubation with washing buffer in each case). The secondary,biotinylated antibody (1 μg/ml in PBS, 100 μl/formulation in each case)was then added; the plates were incubated for 2 hours at 37° C., thenwashed (5 times, 200 μl PBS/formulation in each case) and incubated fora further 2 hours using a streptavidin alkaline phosphatase (AP)conjugate (1 μg/ml in PBS; 200 μl/formulation in each case; Hölzl,Cologne). After a last washing step (5 times, 200 μl PBS/formulation ineach case), the staining reaction took place by adding the enzymesubstrate NBT/BCIP (Boehringer, Mannheim). A staining solution [200 μlNBT/BCIP stock solution to 10 ml of staining buffer (0.1 M tris-buffer,pH 9.5 with 0.05 M MgCl₂, 0.1 M NaCl)] was produced for this purpose and200 μl/formulation was used in each case. After 5-10 min(donor-specific), the calorimetric reaction was stopped by washing theplates with deionised water. The plates were evaluated using an ElispotReader (Biosys 2000, BioSys, Karben).

FIG. 1 shows the results of the IFN-□-elispot for the example of 8donors (6 EBV-positive, 2 EBV-negative probands) after stimulation withurea-denatured BZLF1 polypeptide described above. PBMC of theEBV-negative (JW, BH) and/or B8-negative donors (LD, FN, JW, BH) wereused as negative controls and showed no significant reactivity afterantigen stimulation with urea-denatured BZLF1. In contrast, when usingPBMC from the EBV-positive, HLA B8-positive donors (JU, SD, MB, RE) asignificant to strong reactivity of BZLF1-specific, IFN-β producingcells could be shown. As a result of the depletion of the CD8⁺ T cells,the number of detectable IFN-□ producing ones was reduced significantly,which confirms the presence of BZLF1-specific cytotoxic T cells in thetotal population of peripheral blood lymphocytes.

EXAMPLE 3

Measurement of the virus-specific T-lymphocytes in heparinised wholeblood 10 ml of whole blood was taken from a serologically positive donorin a Heparin Monovette (Sarstedt, Nümbrecht). From this respectively 2ml was pipetted into a sterile Falcon Tube (No. 2059; B D Falcon,Heidelberg). One formulation represents the negative control, in thiscase only the pure monoclonal antibodies against the costimulatorymolecules CD28 and CD49d (Becton Dickinson, Heidelberg) are to be addedin a final concentration of 1 μg/ml. Another formulation is the positivecontrol, in this case a superantigen is pipetted in addition to themonoclonal antibodies CD28 and CD49d. This is the Staphylococcusenterotoxin B (SEB), this is to be added in a final concentration of 1μg/ml. In a third formulation the antigen used for the restimulation isto be added to the whole blood with the monoclonal antibodies. Theconcentration should be titrated out anew for each batch.

In our case, optimal results both with the synthetic peptide and withthe modified protein could be achieved with a final concentration of 10μg/ml. The tube was then incubated in a standard incubator (37° C., 5%CO₂ and H₂O-saturated atmosphere), standing with the lid loose (makinggas exchange possible). After two hours Brefeldin A is added to thesamples so that a final concentration of 10 μg/ml is achieved. Thesamples are thoroughly mixed on a Vortex and placed for another 4 hoursin the incubator.

After a total of 6 hours short-term culture, 11% of the culture volumeis pipetted into ice-cold EDTA solution (EDTA in PBS, 20 mM). Thesamples are briefly vortexed and then incubated for a maximum of 10minutes at room temperature. The samples are then thoroughly vortexedagain so that all the adherent cells are removed from the tube wall.Then at least 9 times the culture volume of FACS lysing solution (BD,Heidelberg) is to be added to the samples. The tubes are then incubatedagain for a maximum of 10 minutes at room temperature. The samples arethen centrifuged at 4° C. for 8 minutes at 340 g. The supernatant iscarefully decanted and the cells are then washed again with 5 mlFACS-buffer (PBS+0.1% w/v NaN₃+1% w/v FCS) and centrifuged again at 340g for 8 minutes. The cells are then resuspended in a small quantity ofFACS buffer and the cells are distributed on the tubes (e.g. Falcon No.2054) for staining. At least 1×10⁶ cells should be present per stainingformulation. The permeabilising solution (PBS+0.1% w/v Saponin) is thenpipetted to the cells in a quantity of 1 ml per 1×10⁶ cells. Theformulation is thoroughly vortexed and incubated for a maximum of 10minutes at room temperature. The cells were then washed with 5 ml FACSbuffer and centrifuged at 340 g for 8 minutes. The cells can then bestained. This takes place in a staining volume of 100 μl. The monoclonalantibodies are then added by pipetting in a ratio of FITC 1:10, PE 1:10,ECD 1:10 and APC 1:100. A typical 4-colour tube would then contain, forexample, the antibodies CD3 FITC, anti-IFN-γ-PE, CD4 ECD and CD8 APC.The staining takes place for at least 30 minutes on ice (4° C.) and inthe dark. The cells are then washed at least twice using 5 ml of FACSbuffer and centrifuged for 8 minutes at 340 g after each washing step.For the measurement the cells are then taken up in at least 200 μl ofFACS buffer. And immediately analysed using a flow cytometer. If thesamples are not to be measured immediately, the cells can be fixed using1% PFA (paraformaldehyde) in FACS buffer. Storage takes place at 4° C.

The results are then evaluated using one of the CellQuest Software(Becton Dickinson, Heidelberg). As an example the figures show theresults for an EBV-seropositive patient showing the HLA-type B8. Afterpermeabilising, the following stainings were carried out:

-   -   Formulation 1: control, unstained    -   Formulation 2: CD3 FITC/IFN-γ PE/CD4 ECD/CD8 APC    -   Formulation 3: CD3 FITC/IL-4 PE/CD4 ECD/CD8 APC    -   Formulation 4: CD3 FITC/CD8 PE/CD4 ECD/CD69 APC    -   Formulation 5: CD3 FITC/CD16 PE/CD8 ECD/CD56 APC    -   Formulation 6: CD3 FITC/perforin PE/CD8 ECD/CD4 APC    -   Formulation 7: CD3 FITC/isotype control PE/CD8 ECD/CD4 APC.

FIGS. 2 a and b show the flow-cytometric analyses of whole blood afterrestimulation with synthetic peptide described above (FIG. 2 a) or afterrestimulation with urea-denatured BZLF1 polypeptide (FIG. 2 b). Shown isan FSC/SSC (Forward Scatter/Side Scatter) dotplot, wherein themeasurement region given by R1 (Region 1) corresponds to the lymphocytepopulation. FIGS. 3 a and b then show the frequency and distribution ofvarious populations of CD3 and CD8-positive lymphocytes in whole bloodafter stimulating with urea-denatured BZLF1 polypeptide (FIG. 3 a) or asynthetic BZLF1 peptide (FIG. 3 a), which contains a known CTL epitope.In this case, it is clear that when stimulating with urea-adjuvatedpolypeptide, significantly more CD3 and CD8 weakly expressing cells canbe retrieved.

FIGS. 4 a and b show the frequency of the peripherally occurringdouble-positive (CD4⁺ and CD8⁺) lymphocyte populations in whole bloodafter stimulating with urea-adjuvated BZLF1 polypeptide (FIG. 4 a) or asynthetic BZLF1 peptide (FIG. 4 b) which contains a known CTL epitope.This figure also clearly shows the advantages of polypeptiderestimulation which allows a considerably better analysis of thedouble-positive T-lymphocytes which can play an important role for theimmune system in virus infections. Furthermore, the population of theweakly CD3 and CD8 expressing cells should be characterised moreaccurately with the staining of the surface markers CD16 and CD56. Thisis because both CD16 and also CD56 are markers which are typical of NKor NKT cells. Since the cells additionally express CD3, the NKT cellscan thus be assigned. FIGS. 5 a and b are dotplots showing the cellpopulations which exhibit expression of the surface markers CD8 andCD56. In both figures (FIGS. 5 a and 5 b), it can be clearly seen thatthe population of CD3⁺ CD8⁺ cells characterised by R3 in FIG. 2, hereshown in blue, expresses CD56 on the cell surface. Thus, the cells shownin R3 express the population of the NKT cells.

Furthermore, with the detection of intracellular IFN-γ it should beshown that the T lymphocytes have been stimulated as part of a Th1immune response and possess cytotoxic properties. In FIGS. 7 a and b thehistogram plots show the interferon-γ expression from variouspopulations of all the lymphocytes registered in R1 after stimulationwith urea-adjuvated BZLF1 polypeptide (FIG. 7 a) or the BZLF1 peptide(FIG. 7 b). Here M1 and M2 are set as markers. M1 designates the regionin which the IFN-γ expression is to be classified as positive.Everything lying to the left thereof is to be explained by non-specificbinding of the anti-IFN-γ antibody inside the cells. The regioncharacterised by M2 shows the IFN-γ expression by the pure population ofCD3⁺CD8⁺ cytotoxic T cells. The difference between the IFN-γ valuesshown in M1 and M2 characterises the IFN-γ production by weak CD8⁺ withNK cell properties.

After restimulating with urea-adjuvated BZLF1 polypeptide (FIG. 7 a), asignificantly increased IFN-γ production from cells of whole blood andan increased stimulation of the population of weakly CD8⁺ cells with NKcell properties can be observed compared to the peptide stimulation. Theexpression of intracellular IFN-γ content was then determined separatelyfor the individual cell populations.

FIGS. 9 a and b show histogram plots giving the IFN-7 expression of thepopulation of CD8⁺ cytotoxic T cells from whole blood shown in R3 afterstimulation with urea-adjuvated BZLF1 polypeptide (FIG. 9 a) or thesynthetic BZLF1 peptide (FIG. 9 a). The markers M1 and M2 are set inaccordance with FIGS. 7 a and b. This figure shows that urea-adjuvatedBZLF1 protein and the synthetic. BZLF1 peptide are equally suitable fordetermining BZLF1-specific cytotoxic T cells. However, the simultaneousreadout of CD8^(dim) cells was only possible after treating the cellswith urea-adjuvated BZLF1 polypeptide.

This phenomenon then becomes clearer in FIGS. 11 a and b. These show, ina histogram plot, the IFN-γ expression of NKT cell population designatedby R4 after stimulating with urea-adjuvated BZLF1 polypeptide (FIG. 11a) or the synthetic BZLF1 peptide (FIG. 11 b). This figure confirms theefficient stimulation of the weakly CD8-positive NKT cell populationafter stimulation with urea-adjuvated BZLF1 polypeptide.

To sum up, it can be shown using the examples that the conventionalmethod corresponding to the prior art for reading out cytotoxic Tlymphocytes by means of restimulation using synthetic peptides actuallyonly allows an analysis of the CD8 highly positive T lymphocytes. Whenusing urea-adjuvated polypeptides however, substantially morepredictions can be made of other cell populations (such as for exampleCD4⁺CD8^(dim) lymphocytes or CD56⁺ NKT cells).

EXAMPLE 4 Induction of a Specific Antibody Response in Rabbits AfterImmunisation with Urea-Adjuvated BZLF1 Polypeptide

In order to test the suitability of urea-adjuvated BZLF-1 polypeptide,30 μg of BZLF1 polypeptide in 8M urea was adjuvated with HuntersTitermax as specified by the manufacturer (Sigma) and administeredintramuscularly to a rabbit. The animal was re-immunised with the sameimmunogen 4 and 8 week as after the basic immunisation. After a further3 weeks, blood was taken from the animal and the serum obtainedtherefrom was tested in different dilutions (1:2000; 1:10000 and1:50000) in the immunoblot for the presence of BZLF1 specificantibodies. For this purpose BZLF1 polypeptide produced recombinantly inE. coli in various concentrations (20 ng, 100 ng and 500 ng) wasseparated in a 12.5% SDS gel and the proteins were then transferred ontonitrocellulose. After the protein transfer the still-free proteinbinding sites on the nitrocellulose were saturated by incubating with a5% solution of skimmed milk powder in TBS (500 mM NaCl, 25 mM Tris pH7.5), shaking slightly. The Blot was then washed four times with TTBSfor at least 10 min before it was incubated for 1 to 12 hours with thecorresponding dilution of the rabbit serum in TBS.

Detection via an enzyme/substrate-mediated colour reaction: After thebinding of the specific antibody, the nitrocellulose filter was againwashed four times with TTBS for 10 min and then shaken for at least 1hour with a suitable dilution of an anti-immunoglobulin coupled withalkali phosphatase (AP) or horseradish peroxidase (HRP).

After a further washing step (four times for 10 min using TTBS), thefilter was incubated with the chromogenic substrates of the alkaliphosphatase (68 μl NBT, 70 μl BCIP in 20 ml AP-buffer: 100 mM NaCl, 50mM MgCl₂, 100 mM Tris (Sambrook et al. 1989)) or horseradish peroxidase(2.5 ml Tris/HCl pH 7.5, 1 spatula tip of 3,3′-diaminobenzidine, 30 μl30% H₂O₂, to 50 ml with H₂O_(bid.)). The resulting enzyme reactionsproduced a brown colour after minutes to hours. The reactions werestopped with H₂O_(bid.).

These studies showed that polypeptide dissolved in 8M urea is suitablefor inducing high-titre polypeptide-specific antibodies. It was shownthat the serum of the experimental animal after three immunisationsstill contains sufficient BZLF1 antibody in a dilution of 1:50000 todetect 100 ng of purified BZLF1 polypeptide in the immunoblot (FIG. 12).The urea contained in the injection solution additionally proved to benon-toxic for the experimental animal.

EXAMPLE 5 Influence of Urea on the Vitality of Purified Population ofPeripheral Blood Mononuclear Cells (PBMC)

In order to test the influence of urea on the cell vitality, PBMC, asdescribed in Example 2, were purified from the heparinised whole bloodof 3 healthy donors (DH, SB, IK) and respectively 1×10⁶ cells in fourreplicates in T-cell medium (RPMI 1640 with 5% human serum, 2 mMglutamine and 100 U/ml penicillin/100 μg/ml streptomycin (all componentsfrom PanSystems, Aidenbach)) was mixed with increasing concentrations ofurea (0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.8 or 1.6 mol/litre) and incubatedin wells of a 48-well microtitre plate for 17 hours in an incubator withhumidified atmosphere and 5% CO₂ gassing at 37° C. An 8M urea stocksolution which was mixed with 2M NaCl and 2 mM DTE was used for theseexperiments. The cells were then resuspended in the cultivationformulation by repeatedly pipetting the medium on and off and the totalnumber of cells and the ratio of the living to dead cells was determinedusing a Neubauer “improved” counting chamber after staining with aTrypan blue solution (0.5% w/v). The results of these investigations aregiven in Table 1. These investigations showed that compared to theuntreated cells, the addition of urea in a concentration range of 0 to0.2 mol/litre results in no significant reduction in the ratio of livingto dead cells in the mixture of peripheral mononuclear cells. When 0.3mol/litre of urea was added, a significantly increased number of deadcells was to be observed; from a urea concentration of 0.4 mol/litre,more dead than living cells were present in the PBMC cultures afterincubation for 17 hours, regardless of the donor. The addition of ureain concentrations above 0.3 mol/litre also resulted in a significantreduction in the total number of cells, which indicates that urea has acytolytic effect at these concentrations. TABLE 1 Influence of the ureaconcentration on the vitality of PBMCs D.H. S.B. I.K. Cell Living/ CellLiving/ Cell Living/ C[urea] number dead number dead number dead 0 1.44× 10⁶ 0.5 1.33 × 10⁶ 34.4 1.5 × 10⁶ 121 mol/ litre 0.05 0.62 × 10⁶ 15.91.03 × 10⁶ 2.2 1.1 × 10⁶ 11.7 mol/ litre 0.1 0.56 × 10⁶ 1.7 1.41 × 10⁶55 1.6 × 10⁶ 41.3 mol/ litre 0.2 0.47 × 10⁶ 6.6 1.94 × 10⁶ 3.6 5.1 × 10⁶30.2 mol/ litre 0.3 n.d. n.d. 0.36 × 10⁶ 1.8 0.7 × 10⁶ 9 mol/ litre 0.40.02 × 10⁶ 1 0.48 × 10⁶ 0.2 0.3 × 10⁶ 0.2 mol/ litre 0.8 0.05 × 10⁶ 0.10.35 × 10⁶ 0.04 2.9 × 10⁶ 0.02 mol/ litre 1.6 0.91 × 10⁶ 0.06 0.76 × 10⁶0 0.4 × 10⁶ 0 mol/ litren.d.: not determined; living/dead cell ratio = 0: that is, exclusivelydead cells in the formulation, for less than 1 there are more dead thanliving cells in the formulation.

EXAMPLE 6 Influence of Urea on the Cytokine Secretion from PurifiedPopulation of Peripheral Blood Mononuclear Cells (PBMC)

Furthermore, it should be clarified by ELIspot analyses whether urea inpopulations of purified PBMCs triggers the secretion of cytokines,especially IFN-γ. For this purpose, 96-well nitrocellulose-coatedmicrotitre plates, as described in detail in Example 2, were coated witha monoclonal anti-human IFN-γ antibody (Hölzl, Cologne) (5 μg/ml in PBS)and incubated overnight at 4° C. The antibody solution was pipetted offand the plates were washed four times using 200 μl PBS/formulation ineach case. In a following step non-specific binding sites were blockedby adding 200 μl of blocking medium in each case (RPMI with 10% FCS)during a one-hour incubation at 37° C.

PBMC of a healthy donor (SB) were taken up in a concentration ofrespectively 2×10⁵ purified PBMC/150 μL in T-cell medium containingdifferent urea concentrations (0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.8, and 1.6mol/litre) (5 replicates each) and the stimulation formulations wereincubated for 24 hours in an incubator with a humidified atmosphere with5% CO₂ gassing at 37° C. An 8M urea stock solution which was mixed with2M NaCl and 2 mM DTE was used for these experiments. The cell suspensionwas then drawn off and any cells still adhering were removed by washingsix times (in each case 200 μl PBS with 0.1% Tween 20, 3 min incubationwith washing buffer in each case). The secondary biotinylated antibody(1 μg/ml in PBS, 100 μl/formulation in each case) was then added; theplates were incubated for 2 hours at RT, then washed (6 times, 200 μlPBS/formulation in each case) and incubated for 1 hour with astreptavidin alkaline phosphatase (AP) conjugate (1 μg/ml in PBS; 100μl/formulation in each case; Hölzl, Cologne). After a last washing step(6 times 200 μl PBS/formulation in each case), the colour reaction tookplace by adding the enzyme substrate NBT/BCIP (Boehringer, Mannheim).For this purpose a staining solution was prepared [200 μl NBT/BCIP-stocksolution to 10 ml staining buffer (0.1 M tris-buffer, pH 9.5 with 0.05 MMgCl₂, 0.1 M NaCl)] and 100 μl/formulation was used in each case.

After 10 min the calorimetric reaction was stopped by washing the plateswith deionised water (Biosys 2000, BioSys, Karben).

Table 2 shows the results of the IFN-γ-Elispot for the example of adonor after stimulation using the urea concentrations describedpreviously. These investigations showed that after adding differentconcentrations of urea, no significantly increased release of IFN-γ isinduced from cultures of peripheral blood mononuclear cells. TABLE 2Influence of urea concentration on IFN-γ secretion from PBMCs S.B.Spots/2 × 10⁶ Standard c[urea] cells deviation   0 mol/litre 18.4 5.460.05 mol/litre 26.2 12.9  0.1 mol/litre 29.6 9.99  0.2 mol/litre 26.416.5  0.3 mol/litre 11.0 4.15  0.4 mol/litre 18.2 3.12  0.8 mol/litre14.6 3.56  1.6 mol/litre 13.4 1.74

EXAMPLE 7 Dose Dependence of IFN-γ Secretion After Stimulation withUrea-Treated BZLF-1 Protein

The suitability of denatured polypeptides and polypeptides present inurea solution for inducing an epitope presentation on MHC class I and IIproteins as well as for simultaneously detecting polypeptide-specificCD4⁺ and CD8⁺ T cells in mixed APC/lymphocyte cultures was tested usinga very thoroughly characterised model system. This is based on theobservation that all HLA B8-positive, EBV-positive probands possess CD8⁺cytotoxic T cells which recognize a specific epitope (RAKFKQLL; aminoacid 190-197) inside the EBV protein BZLF1 (Bogedain et al. (1995); J.Virol. 69:4872; Pepperl et al., (1998) J. Virol. 72:8644). In order totest the optimal protein concentration to detect BZLF-1 specific T cellresponses, purified peripheral blood lymphocytes (PBMC) from variousEBV-negative, HLA B8-positive (donor 1), or EBV-positive, HLAB8-negative (donors 2,3) or EBV-positive, HLA B8-positive probands(donors 4-7) were incubated with various concentrations ofurea-adjuvated BZLF-1 proteins and the IFN-γ secretion after 17 hourswas determined using the ELIspot method.

For this purpose, nitrocellulose-coated 96-well microtitre plates, asdescribed in detail in Example 2, were coated with a monoclonal,anti-human IFN-γ antibody (Hölzl, Cologne) (5 μg/ml in PBS) andincubated overnight at 4° C.

The antibody solution was pipetted off and the plates were washed fourtimes using 200 μl PBS/formulation in each case. In a following stepnon-specific binding sites were blocked by adding 200 μl of blockingmedium (RPMI with 10% FCS) in each case during a one-hour incubation at37° C.

PBMCs from the seven probands in a concentration of 2×10⁵ purifiedPBMC/150 μL in each case, were then taken up in T cell medium containingdifferent concentrations of urea-adjuvated BZLF-1 protein (0.5 μg/ml, 2μg/ml, 5 μg/ml, 20 μg/ml) (5 replicates each) and the stimulationformulations were incubated for 24 hours in an incubator with humidifiedatmosphere with 5% CO₂ gassing at 37° C. BZLF-1 protein which waspresent with a concentration of 1 mg/ml in an 8 molar urea with 2M NaCland 2 mM DTE was used for these experiments. The cell suspension wasthen drawn off and any cells still adhering were removed by washing 6times (200 μl PBS with 0.1% Tween 20 in each case, 3 min incubation withwashing buffer in each case). The secondary biotinylated antibody wasthen added (1 μg/ml in PBS, 100 μl/formulation in each case) was thenadded; the plates were incubated for 2 hours at RT, then washed (6 times200 μl PBS/formulation in each case) and incubated for 1 hour using astreptavidin alkaline phosphatase (AP) conjugate (1 μg/ml in PBS; 100μl/formulation in each case; Hölzl, Cologne). After a last washing step(6 times 200 μl PBS/formulation in each case), the colour reaction tookplace by adding the enzyme substrate NBT/BCIP (Boehringer, Mannheim).For this purpose a staining solution was prepared [200 μl NBT/BCIP-stocksolution to 10 ml staining buffer (0.1 M tris-buffer, pH 9.5 with 0.05 MMgCl₂, 0.1 M NaCl)] and 100 μl/formulation was used in each case.

After 5-10 min (donor-specific) the calorimetric reaction was stopped bywashing the plates with deionised water. The plates were evaluated on anElispot reader (Biosys 2000, BioSys, Karben). TABLE 3 Relationship ofthe BZLF1 protein concentration to the urea concentration c[BZLF1]c[urea]  5 μg/ml 0.04 mol/litre 10 μg/ml 0.08 mol/litre 20 μg/ml 0.16mol/litre 30 μg/ml 0.24 mol/litreRelationship of the BZLF1 protein concentration to the ureaconcentration using a stock solution containing 1 μg/μl BZLF1 protein in8 mol/litre urea, 2 mol/litre NaCl, 2 mmol/litre 1,4-dithioerythritol(DTE).

The results of this experiment are shown in FIG. 13. The investigationsshowed that the BPMCs of 2 out of the 4 tested HLA-B8-positive,EBV-positive donors at all the BZLF-1 concentrations tested showed asignificantly increased number of IFN-γ producing BZLF-1-specific Tcells compared to the negative controls (donors 1-3). In theseexperiments low concentrations of the urea-adjuvated BZLF-1 protein werealready sufficient to detect a significantly increased number ofBZLF-1-specific IFN-γ producing T cells compared to the negativecontrols (donors 1-3) in 3 out of 4 HLA-B8-positive, EBV-positivedonors. The optimal stimulation of interferon-γ production was to beobserved after stimulation with 5 or 20 μg/ml urea-adjuvated BZLF-1protein specific to the donor. After stimulating PBMCs of controlprobands 1 and 3 with urea-adjuvated BZLF-1 protein in concentrations of2 to 20 μg/ml, a small number of IFN-γ secreting PBMCs could bedetected. These reactivities are possibly based on the stimulation ofCD8⁺ T cell which are directed against hitherto unknown target epitopeswithin the BZLF-1 protein. Furthermore, the observed spot may come fromBZLF-1-specific CD4⁺ cells which are excited to IFN-γ production by thestimulation with urea-adjuvated BZLF-1 protein.

EXAMPLE 8 Time Behaviour of IFN-γ Secretion After Stimulation withUrea-Treated BZLF-1 Protein

In order to test the optimal stimulation time to detect BZLF-1-specificT cell responses, purified peripheral blood lymphocytes (PBMC) fromvarious EBV-negative, HLA B8-positive (donor 1), or EBV-positive, HLAB8-negative (donors 2,3) or EBV-positive, HLA B8-positive probands(donors 4-7) were incubated for varying times using 10 μg/mlurea-adjuvated BZLF-1 proteins in each case and the IFN-γ secretion wasdetermined using the ELIspot method.

For this purpose nitrocellulose-coated 96-well microtitre plates, asalready described in detail, were coated with a monoclonal, anti-humanIFN-γ antibody and non-specific binding sites were blocked by addingblocking medium (RPMI with 10% FCS). Then PBMCs of the seven probands ina concentration of 2×10⁵ purified PBMC/150 μL in each case were taken upin T-cell medium using 10 μg/ml urea-adjuvated BZLF-1 protein (5replicates in each case) and the stimulation formulations were incubatedfor 2, 8, 16 or 24 hours in an incubator with humidified atmosphere with5% CO₂ gassing at 37° C. BZLF-1 protein which was present in aconcentration of 1 mg/ml in an 8 molar urea with 2M NaCl and 2 mM DTEwas used for these experiments. The IFN-γ ELIspot Assay was carried outas described in detail in the preceding example.

The results of this experiment are shown in FIG. 14. The investigationsshowed that after incubation for 8 hours all HLA-B8-positive,EBV-positive donors (donors 4-7) show a significantly increased numberof IFN-γ producing BZLF-1-specific T cells compared with all “controlprobands” (donors 1-3). The maximum number of IFN-γ producing cellscould be observed after 16 to 24 hours depending on the donor.

1. A method for polypeptide transfer in APC cells, comprising thefollowing steps: a) incubating polypeptides with a urea solution, b)incubating cells with the polypeptides present in the urea solution. 2.A method for the detection of polypeptide-specific immune cells,comprising the following steps: a) incubating polypeptides with a ureasolution, b) incubating APC-containing cell cultures or body fluids withthe polypeptides present in the urea solution, c) incubating theAPC-containing cell cultures or body fluids obtained according to stepb) with immune cells or immune-cell-containing body fluids, d)simultaneously detecting and/or quantifying various subtypes of immunecells which are specific against the polypeptides from step a).
 3. Themethod according to claim 1, wherein the urea solution in step a) has aconcentration in the range of about 0.01 mol/litre to about 10mol/litre.
 4. The method according to claim 1, wherein the polypeptidesin step a) have a concentration in the range of about 0.01 μg/μl toabout 50 μg/μl.
 5. The method according to claim 1, wherein thepolypeptides in step b) have a concentration in the range of about 0.1μg to about 200 μg for approximately 10⁶ cells.
 6. The method accordingto claim 1, wherein the urea concentration in step b) has a finalconcentration in the range of about 0.001 to about 0.3 mol/litre or lessthan 0.001 mol/litre or more than 0.3 mol/litre.
 7. The method accordingto claim 6, wherein the urea solution additionally contains NaCl with aconcentration in the range of about 0.25 mmol/litre to about 75mmol/litre and/or DTE with a concentration in the range of about 0.25mmol/litre to about 75 nmol/litre.
 8. The method according to claim 2,wherein the APC-containing cell culture is a PBMC population(leukapheresate), isolated monocytic cells or a separated APC populationand the APC-containing body fluid is whole blood or liquor.
 9. Themethod according to claim 8, wherein the separated APC population hasdendritic cells (Langerhans cells), monocytes, macrophages or B cells.10. The method according to claim 2, wherein the immune cells are Tcells or other immunological cell populations or a mixture thereof andthe immune-cell-containing body fluids are whole blood or liquor. 11.The method according to claim 10, wherein the T cells are CD4⁺ T cells,CD8⁺ T cells, CD4⁺CD8^(dim) T cells or CD4⁺CD25⁺ suppressor T cells andthe immunological cell populations are CD56⁺CD8⁺, CD56⁻CD57⁺CD8⁺ NKTcells or CD56⁺ NK cells.
 12. The method according to claim 2, whereinthe detection and/or the quantification is carried out by detection ofspecific surface markers for immune cells and IFNγ, IL4 or IL5.
 13. Themethod according to claim 2, wherein the detection is carried out usingFACS, ELISA or Elispot methods or biosensors.
 14. Use of the cellsobtained according to claim 1 for research, diagnosis or treatment andprevention of diseases in animals and humans.
 15. Cells obtainedaccording to a method comprising the steps: a) incubating polypeptideswith a urea solution, b) incubating cells with the polypeptides presentin the urea solution.
 16. The method according to claim 2, wherein theurea solution in step a) has a concentration in the range of about 0.01mol/litre to about 10 mol/litre.
 17. The method according to claim 2,wherein the polypeptides in step a) have a concentration in the range ofabout 0.01 μg/μl to about 50 μg/μl.
 18. The method according to claim 1,wherein the polypeptides in step b) have a concentration in the range ofabout 0.1 μg to about 200 μg for approximately 10⁶ cells.
 19. The methodaccording to claim 2, wherein the urea concentration in step b) has afinal concentration in the range of about 0.001 to about 0.3 mol/litreor less than 0.001 mol/litre or more than 0.3 mol/litre.